Molecular Basis for the Reciprocal Regulation of P. aeruginosa Virulence Factors by the Signaling Kinase RetS Abstract The nosocomial pathogen Pseudomonas aeruginosa causes acute and persistent, chronic infections using distinct virulence mechanisms. The sensor signaling kinase RetS is a major player in regulating these virulence mechanisms by inhibiting the signaling kinase GacS. The unique molecular mechanism underlying this inhibition has not been determined. Our preliminary data refute the original hypothesis that RetS interferes with GacS homo-dimerization. Here we propose to test an alternative model wherein RetS directly blocks the active and phosphorylation sites of GacS. The second part of the proposed work seeks to determine how this inhibition is relieved when the bacterium enters biofilm mode to cause an expression of entirely different set of virulence factors. We hypothesize that binding of the exopolysaccharide PSL to the sensory domain of RetS causes RetS autophosphorylation, which in turn triggers the release of GacS. Structural studies and a series of in vitro and in vivo assays are proposed to address these important biological questions. In addition, the use of the NarX sensory domain to regulate the kinase activity of RetS constitutes an important extension of previous applications. We are controlling an enzyme that does not have a HAMP domain normally required for NarX signaling. If our approach is successful it could be applied to studies of many more orphan signaling kinases.